Prostaglandin derivatives

ABSTRACT

Prostaglandin nitroderivatives having improved pharmacological activity and enhanced tolerability are described. They can be employed for the treatment of glaucoma and ocular hypertension.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is based upon and claims the benefit of priority fromEuropean Patent Application No. 04100001.9 filed on Jan. 5, 2004.

BACKGROUND OF THE INVENTION

The present invention relates to new prostaglandin derivatives. Moreparticularly, the present invention relates to prostaglandinnitrooxyderivatives, pharmaceutical compositions containing them andtheir use as drugs for treating glaucoma and ocular hypertension.

Glaucoma is optic nerve damage, often associated with increasedintraocular pressure (IOP), that leads to progressive, irreversible lossof vision.

Almost 3 million people in the United States and 14 million peopleworldwide have glaucoma; this is the third leading cause of blindnessworldwide.

Glaucoma occurs when an imbalance in production and drainage of fluid inthe eye (aqueous humor) increases eye pressure to unhealthy levels.

It is known that elevated IOP can be at least partially controlled byadministering drugs which either reduce the production of aqueous humorwithin the eye or increase the fluid drainage, such as beta-blockers,α-agonists, cholinergic agents, carbonic anhydrase inhibitors, orprostaglandin analogs.

Several side effects are associated with the drugs conventionally usedto treat glaucoma.

Topical beta-blockers show serious pulmonary side effects, depression,fatigue, confusion, impotence, hair loss, heart failure and bradycardia.

Topical α-agonists have a fairly high incidence of allergic or toxicreactions; topical cholinergic agents (miotics) can cause visual sideeffects.

The side effects associated with oral carbonic anhydrase inhibitorsinclude fatigue, anorexia, depression, paresthesias and serumelectrolyte abnormalities (The Merck Manual of Diagnosis and Therapy,Seventeenth Edition, M. H. Beers and R. Berkow Editors, Sec. 8, Ch.100).

Finally, the topical prostaglandin analogs (bimatoprost, latanoprost,travoprost and unoprostone) used in the treatment of glaucoma, canproduce ocular side effects, such as increased pigmentation of the iris,ocular irritation, conjunctival hyperaemia, iritis, uveitis and macularoedema (Martindale, Thirty-third edition, p. 1445).

U.S. Pat. No. 3,922,293 describes monocarboxyacylates of prostaglandinsF-type and their 15β isomers, at the C-9 position, and processes forpreparing them; U.S. Pat. No. 6,417,228 discloses 13-aza prostaglandinshaving functional PGF_(2α) receptor agonist activity and their use intreating glaucoma and ocular hypertension.

WO 90/02553 discloses the use of prostaglandins derivatives of PGA, PGB,PGE and PGF, in which the omega chain contains a ring structure, for thetreatment of glaucoma or ocular hypertension.

WO 00/51978 describes novel nitrosated and/or nitrosylatedprostaglandins, in particular novel derivatives of PGE₁, novelcompositions and their use for treating sexual dysfunctions.

U.S. Pat. No. 5,625,083 discloses dinitroglycerol esters ofprostaglandins which may be used as vasodilators, antihypertensivecardiovascular agents or bronchodilators.

U.S. Pat. No. 6,211,233 discloses compounds of the general formulaA-X₁—NO₂, wherein A contains a prostaglandin residue, in particularPGE₁, and X₁ is a bivalent connecting bridge, and their use for treatingimpotence.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide new derivatives ofprostaglandins able not only to eliminate or at least reduce the sideeffects associated with these compounds, but also to possess an improvedpharmacological activity. It has been surprisingly found thatprostaglandin nitroderivatives have a significantly improved overallprofile as compared to native prostaglandins both in terms of widerpharmacological activity and enhanced tolerability. In particular, ithas been recognized that the prostaglandin nitroderivatives of thepresent invention can be employed for treating glaucoma and ocularhypertension. The compounds of the present invention are indicated forthe reduction of intraocular pressure in patients with open-angleglaucoma or with chronic angle-closure glaucoma who underwent peripheraliridotomy or laser iridoplasty.

DETAILED DESCRIPTION OF THE INVENTION

An object of the present invention is, therefore, prostaglandinnitroderivatives of general formula (I) and pharmaceutically acceptablesalts or stereoisomers thereof

R—X—Y—ONO₂  (I)

wherein R is the prostaglandin residue of formula (II):

whereinthe symbol

represents a single bond or a double bond;L is selected from the following groups:

X is —O—, —S— or —NH—;

Y is a bivalent radical having the following meaning:a)

-   -   straight or branched C₁-C₂₀ alkylene, preferably C₁-C₁₀, being        optionally substituted with one or more of the substituents        selected from the group consisting of: halogen atoms, hydroxy,        —ONO₂ or T, wherein T is    -   —OC(O)(C₁-C₁₀ alkyl)-ONO₂ or —O(C₁-C₁₀ alkyl)-ONO₂;    -   cycloalkylene with 5 to 7 carbon atoms into cycloalkylene ring,        the ring being optionally substituted with side chains T₁,        wherein T₁ is straight or branched C₁-C₁₀ alkyl, preferably CH₃;

wherein n is an integer from 0 to 20, and n¹ is an integer from 1 to 20;

wherein

X₁=—OCO— or —COO— and R² is H or CH₃;

Z is —(CH)_(n) ¹— or the bivalent radical defined above under b)n¹ is as defined above and n² is an integer from 0 to 2;

wherein:Y¹ is —CH₂—CH₂—(CH₂)_(n) ²—; or —CH═CH—(CH₂)_(n) ²—;Z is —(CH)_(n) ¹— or the bivalent radical defined above under b)n¹, n², R² and X₁ are as defined above;

wherein:n¹ and R² are as defined above, R is H or —COCH₃;with the proviso that when Y is selected from the bivalent radicalsmentioned under b)-f), the terminal —ONO₂ group is bound to —(CH₂)_(n)¹;

wherein X₂ is —O— or —S—, n³ is an integer from 1 to 6, preferably from1 to 4, R² is as defined above;

wherein:n⁴ is an integer from 0 to 10;n⁵ is an integer from 1 to 10;R⁴, R⁵, R⁶, R⁷ are the same or different, and are H or straight orbranched C₁-C₄ alkyl, preferably R⁴, R⁵, R⁶, R⁷ are H;wherein the —ONO₂ group is linked to

wherein n⁵ is as defined above;Y² is an heterocyclic saturated, unsaturated or aromatic 5 or 6 membersring, containing one or more heteroatoms selected from nitrogen, oxygen,sulfur,and is selected from

The term “C₁-C₂₀ alkylene” as used herein refers to branched or straightchain C₁-C₂₀ hydrocarbon, preferably having from 1 to 10 carbon atomssuch as methylene, ethylene, propylene, isopropylene, n-butylene,pentylene, n-hexylene and the like.

The term “C₁-C₁₀ alkyl” as used herein refers to branched or straightchain alkyl groups comprising one to ten carbon atoms, including methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl,octyl and the like.

The term “cycloalkylene” as used herein refers to ring having from 5 to7 carbon atoms including, but not limited to, cyclopentylene,cyclohexylene optionally substituted with side chains such as straightor branched (C₁-C₁₀)-alkyl, preferably CH₃.

The term “heterocyclic” as used herein refers to saturated, unsaturatedor aromatic 5 or 6 members ring, containing one or more heteroatomsselected from nitrogen, oxygen, sulphur, such as for example pyridine,pyrazine, pyrimidine, pyrrolidine, morpholine, imidazole and the like.

As stated above, the invention includes also the pharmaceuticallyacceptable salts of the compounds of formula (I) and stereoisomersthereof.

Examples of pharmaceutically acceptable salts are either those withinorganic bases, such as sodium, potassium, calcium and aluminiumhydroxides, or with organic bases, such as lysine, arginine,triethylamine, dibenzylamine, piperidine and other acceptable organicamines.

The compounds according to the present invention, when they contain inthe molecule one salifiable nitrogen atom, can be transformed into thecorresponding salts by reaction in an organic solvent such asacetonitrile, tetrahydrofuran with the corresponding organic orinorganic acids.

Examples of organic acids are: oxalic, tartaric, maleic, succinic,citric acids. Examples of inorganic acids are: nitric, hydrochloric,sulphuric, phosphoric acids. Salts with nitric acid are preferred.

The compounds of the invention which have one or more asymmetric carbonatoms can exist as optically pure enantiomers, pure diastereomers,enantiomers mixtures, diastereomers mixtures, enantiomer racemicmixtures, racemates or racemate mixtures.

Within the scope of the invention are also all the possible isomers,stereoisomers and their mixtures of the compounds of formula (I),including mixtures enriched in a particular isomer.

Preferred compounds of formula (I) are those wherein R, L, X are asdefined in claim 1 and Y is a bivalent radical having the followingmeaning:

a)

-   -   straight or branched C₁-C₂₀ alkylene, being optionally        substituted with one or more of the substituents selected from        the group consisting of: halogen atoms, hydroxy, —ONO₂ or T,        wherein T is    -   —OC(O)(C₁-C₁₀ alkyl)-ONO₂ or —O(C₁-C₁₀ alkyl)-ONO₂;    -   cycloalkylene with 5 to 7 carbon atoms into cycloalkylene ring,        the ring being optionally substituted with side chains T₁,        wherein T₁ is straight or branched C₁-C₁₀ alkyl;

wherein n is an integer from 0 to 20, and n¹ is an integer from 1 to 20;

wherein:n¹ is as defined above and n² is an integer from 0 to 2;

X₁=—OCO— or —COO— and R² is H or CH₃;

wherein:n¹, n², R² and X₁ are as defined above;Y¹ is —CH₂—CH₂— or —CH═CH—(CH₂)_(n) ²—;

wherein:n¹ and R² are as defined above, R³ is H or —COCH₃;with the proviso that when Y is selected from the bivalent radicalsmentioned under b)-f), the —ONO₂ group is bound to —(CH₂)_(n) ¹;

wherein X₂ is —O— or —S—, n³ is an integer from 1 to 6 and R² is asdefined above;

wherein:n⁴ is an integer from 0 to 10;n⁵ is an integer from 1 to 10;R⁴, R⁵, R⁶, R⁷ are the same or different, and are H or straight orbranched C₁-C₄ alkyl;wherein the —ONO₂ group is linked to

wherein n⁵ is as defined above;Y² is an heterocyclic saturated, unsaturated or aromatic 5 or 6 membersring, containing one or more heteroatoms selected from nitrogen, oxygen,sulfur,and is selected from

Preferred compounds of formula (I) are those wherein

the prostaglandin residue R is selected from the group consisting oflatanoprost, travoprost, unoprostone and cloprostenol, preferably R islatanoprost.X is preferably —O— or —S—;

A preferred group of compounds of general formula (I) are those whereinY is a bivalent radical having the following meaning:

a)

-   -   straight or branched C₂-C₆ alkylene, being optionally        substituted with —ONO₂ or T, wherein T is as above defined;

wherein n is an integer from 0 to 5, and n¹ is an integer from 1 to 5;

wherein X₂ is —O— or —S—, n³ is 1, R² is as defined above.

Most preferred meanings of Y are:

a) branched C₂-C₆ alkylene or straight or branched C₂-C₆ alkylene beingoptionally substituted with —ONO₂ or T, wherein T is as defined in claim1;

wherein n is 0, and n¹ is 1.

wherein X₂ is —O— or —S—, n³ is 1, R² is hydrogen;

Another preferred group of compounds of general formula (I) are thosewherein Y is a bivalent radical having the following meaning:

wherein

X₁=—OCO— or —COO— and R² is H or CH₃;

Z is —(CH)_(n) ¹— or the bivalent radical defined above under b) whereinn is an integer from 0 to 5;n¹ is an integer from 1 to 5 and n² is an integer from 0 to 2;

wherein:Y¹ is —CH₂—CH₂—(CH₂)_(n) ²—; or —CH═CH—(CH₂)²—;Z is —(CH)_(n) ¹— or the bivalent radical defined above under b)n¹, n², R² and X₁ are as defined above;

wherein:n¹ and R² are as defined above, R³ is H or COCH₃;with the proviso that when Y is selected from the bivalent radicalsmentioned under b)-f), the —ONO₂ group is bound to —(CH₂)_(n) ¹;

wherein:n⁴ is an integer from 0 to 3;n⁵ is an integer from 1 to 3;R⁴, R⁵, R⁶, R⁷ are the same and are H;and wherein the —ONO₂ group is linked to

Y² is a 6 member saturated, unsaturated or aromatic heterocyclic ring,containing one or two atoms of nitrogen and selected for example from

The following are preferred compounds according to the presentinvention:

As mentioned above, objects of the present invention are alsopharmaceutical compositions containing at least a compound of thepresent invention of formula (I) together with non toxic adjuvantsand/or carriers usually employed in the pharmaceutical field.

The preferred route of administration is topical.

The compounds of the present invention can be administered as solutions,suspensions or emulsions (dispersions) in an ophthalmically acceptablevehicle. The term “ophthalmically acceptable vehicle” as used hereinrefers to any substance or combination of substances which arenon-reactive with the compounds and suitable for administration topatient.

Preferred are aqueous vehicles suitable for topical application to thepatient's eyes.

Other ingredients which may be desirable to use in the ophthalmiccompositions of the present invention include antimicrobials,preservatives, co-solvents, surfactants and viscosity building agents.

The invention also relates to a method for treating glaucoma or ocularhypertension, said method consisting in contacting an effectiveintraocular pressure reducing amount of a composition with the eye inorder to reduce eye pressure and to maintain said pressure on a reducedlevel.

The doses of prostaglandin nitroderivatives can be determined bystandard clinical techniques and are in the same range or less thanthose described for the corresponding underivatized, commerciallyavailable prostaglandin compounds as reported in the: Physician's DeskReference, Medical Economics Company, Inc., Oradell, N.J., 58^(th) Ed.,2004; The pharmacological basis of therapeutics, Goodman and Gilman, J.G. Hardman, L. e. Limbird, Tenth Ed.

The compositions contain 0.1-0.30 μg, especially 1-10 μg, perapplication of the active compound.

The treatment may be advantageously carried out in that one drop of thecomposition, corresponding to about 30 μl, is administered about 1 to 2times per day to the patient's eye.

It is further contemplated that the compounds of the present inventioncan be used with other medicaments known to be useful in the treatmentof glaucoma or ocular hypertension, either separately or in combination.For example the compounds of the present invention can be combined with(i) beta-blockers, such as timolol, betaxolol, levobunolol and the like(see U.S. Pat. No. 4,952,581); (ii) carbonic anhydrase inhibitors, suchas brinzolamide; (iii) adrenergic agonists including clonidinederivatives, such as apraclonidine or brimonidine (see U.S. Pat. No.5,811,443. Also contemplated is the combination with nitrooxyderivatives of the above reported compounds, for example nitrooxyderivatives of beta-blockers such as those described in U.S. Pat. No.6,242,432.

The compounds of the present invention can be synthesized as follows.

Synthesis Procedure

The compounds of general formula (I) as above defined, can be obtained:

i) by reacting a compound of formula (III)

whereinL is as above defined; P is H or a hydroxylic protecting group such assilyl ethers, such as trimethylsilyl, tert-butyl-dimethylsilyl or acetyland those described in T. W. Greene “Protective groups in organicsynthesis”, Harvard University Press, 1980, 2^(nd) edition, p. 14-118; Wis —OH, Cl, or —OC(O)R₁ wherein R₁ is a linear or branched C₁-C₅ alkyl;with a compound of formula (IV) Z-Y-Q wherein Y is as above defined, Zis HX or Z₁, being X as above defined and Z₁ selected from the groupconsisting of:chlorine, bromine, iodine, mesyl, tosyl;

Q is —ONO₂ or Z₁ and

ii) when Q is Z₁, by converting the compound obtained in the step i)into nitro derivative by reaction with a nitrate source such as silvernitrate, lithium nitrate, sodium nitrate, potassium nitrate, magnesiumnitrate, calcium nitrate, iron nitrate, zinc nitrate ortetraalkylammonium nitrate (wherein alkyl is C₁-C₁₀ alkyl) in a suitableorganic solvent such as acetonitrile, tetrahydrofurane, methyl ethylketone, ethyl acetate, DMF, the reaction is carried out, in the dark, ata temperature from room temperature to the boiling temperature of thesolvent. Preferred nitrate source is silver nitrate and

iii) optionally deprotecting the compounds obtained in step i) or ii) asdescribed in T. W. Greene “Protective groups in organic synthesis”,Harvard University Press, 1980, 2^(nd) edition, p. 68-86. Fluoride ionis the preferred method for removing silyl ether protecting group.

-   -   The reaction of a compound of formula (III) wherein W=—OH, P and        X₁ are as above defined, with a compound of formula (IV) wherein        Y and Q are as above defined, Z is HX may be carried out in        presence of a dehydrating agent as        dicyclohexylcarbodiimide (DCC) or        N′-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride        (EDAC) and a catalyst, such as N,N-dimethylamino pyridine        (DMAP). The reaction is carried out in an inert organic solvent        dry such as N,N′-dimethylformamide, tetrahydrofuran, benzene,        toluene, dioxane, a polyhalogenated aliphatic hydrocarbon at a        temperature from −20° C. and 40° C. The reaction is completed        within a time range from 30 minutes to 36 hours.    -   The compounds of formula (III) wherein W=—OH and P═H are        commercially available;    -   The compounds of formula (III) wherein W=—OH and P is a        hydroxylic protecting group may be prepared from the        corresponding compounds wherein P═H as well known in the art,        for example as described in T. W. Greene “Protective groups in        organic synthesis”, Harvard University Press, 1980, 2^(nd)        edition, p. 14-118.    -   The reaction of a compound of formula (III) wherein W=—OC(O)R₁        wherein R₁ is as above defined and P═H or a hydroxylic        protecting group, with a compound of formula (IV) wherein Y is        as above defined, Z is —OH and Q is —ONO₂ may be carried out in        presence of a catalyst, such as N,N-dimethylamino pyridine        (DMAP). The reaction is carried out in an inert organic solvent        such as N,N′-dimethylformamide, tetrahydrofuran, benzene,        toluene, dioxane, a polyhalogenated aliphatic hydrocarbon at a        temperature from −20° C. and 40° C. The reaction is completed        within a time range from 30 minutes to 36 hours.    -   The compounds of formula (III) wherein W=—OC(O)R₁ and P═H may be        obtained from the corresponding acids wherein W=—OH by reaction        with a chloroformate such as isobutylchloroformate,        ethylchloroformate in presence of a non-nucleophilic base such        as triethylamine in an inert organic solvent such as        N,N′-dimethylformamide, tetrahydrofuran, a polyhalogenated        aliphatic hydrocarbon at a temperature from −20° C. and 40° C.        The reaction is completed within a time range from 1 to 8 hours.    -   The reaction of a compound of formula (III) wherein W=—OH and        P═H, with a compound of formula (IV) wherein Y is as above        defined, Z is Z₁ and Q is —ONO₂ may be carried out in presence        of a organic base such as 1,8-diazabiciclo[5.4.0]undec-7-ene        (DBU), N,N-diisopropylethylamine, diisopropylamine or inorganic        base such as alkaline-earth metal carbonate or hydroxide,        potassium carbonate, cesium carbonate, in an inert organic        solvent such as N,N′-dimethylformamide, tetrahydrofuran,        acetone, methyl ethyl ketone, acetonitrile, a polyhalogenated        aliphatic hydrocarbon at a temperature from −20° C. and 40° C.,        preferably from 5° C. to 25° C. The reaction is completed within        a time range from 1 to 8 hours. When Z₁ is chosen among chlorine        or bromine the reaction is carried out in presence an iodine        compound such as KI.    -   The reaction of a compound of formula (III) wherein W=Cl and P        is as above defined, with a compound of formula (IV) wherein Y        is as above defined, Z is —OH and Q is —ONO₂ may be carried out        in presence of a of a organic base such as N,N-dimethylamino        pyridine (DMAP), triethylamine, pyridine. The reaction is        carried out in an inert organic solvent such as        N,N′-dimethylformamide, tetrahydrofuran, benzene, toluene,        dioxane, a polyhalogenated aliphatic hydrocarbon at a        temperature from −20° C. and 40° C. The reaction is completed        within a time range from 30 minutes to 36 hours.    -   The compounds of formula (III) wherein W=Cl may be obtained from        the corresponding acids wherein W=—OH by reaction with a thionyl        or oxalyl chloride, halides of P^(III) or P^(V) in solvents        inert such as toluene, chloroform, DMF.    -   The compounds of formula HO—Y—ONO₂, wherein Y is as above        defined can be obtained as follows. The corresponding diol        derivative, commercially available, or synthesized by well known        reactions, is converted in HO—Y-Z₁, wherein Z₁ is as above        defined, by well known reactions, for example by reaction with        thionyl or oxalyl chloride, halides of P^(III) or P^(V), mesyl        chloride, tosyl chloride in solvents inert such as toluene,        chloroform, DMF, etc. The conversion to the nitro derivative is        carried out as above described. Alternatively the diol        derivative can be nitrated by reaction with nitric acid and        acetic anhydride in a temperature range from −50° C. to 0° C.        according to methods well known in the literature.    -   The compounds of formula Z₁-Y—ONO₂, wherein Y and Z₁ are as        above defined can be obtained from the halogen derivative        Z₁-Y-Hal, commercially available or synthesized according to        methods well known in the literature, by conversion to the nitro        derivative as above described.    -   The compounds of formula H—X—Y-Z₁, wherein X, Y and Z₁ are as        above defined can be obtained from the hydroxyl derivative        H—X—Y—OH, commercially available or synthesized according to        methods well known in the literature, by well known reactions,        for example by reaction with thionyl or oxalyl chloride, halides        of P^(III) or P^(V), mesyl chloride, tosyl chloride in solvents        inert such as toluene, chloroform, DMF, etc.

The following examples are to further illustrate the invention withoutlimiting it.

EXAMPLE 1 Synthesis of[1R-[1α(Z),2α(R*),3α,5α]]-7-[3,5-dihydroxy-2-(3-hydroxy-5-phenylpentyl)cyclopentyl]-5-heptenoicacid 4-(nitrooxy)butyl ester (compound 1)

II EXPERIMENTAL II.1 Preparation of 4-bromobutanol

Tetrahydrofuran (12.5 g-173 mmol) was charged under nitrogen in areactor cooled to 5-10° C. Hydrogen bromide (7.0 g-86.5 mmol) was thenadded slowly and the reaction medium was stirred over a period of 4.5hours at 5-10° C. The mixture was diluted with 22.5 g of cold water andthe pH of this solution was adjusted to pH=5-7 by adding 27.65% sodiumhydroxide (2.0 g) keeping the temperature at 5-10° C. The solution wasthen extracted twice with dichloromethane (13.25 g). The combinedorganic phases were washed with 25% brine (7.5 g), adjusted to pH=6-7with 27.65% sodium hydroxide and dried over magnesium sulfate.Dichloromethane was distilled off and crude 4-bromobutanol (10.3 g-66.9mmol) was obtained in a yield of about 77%.

II.2 Preparation of 4-bromobutyl nitrate

In reactor cooled to −5 to 5° C., nitric acid fuming (8.5 g-135 mmol)was slowly added to a solution of 98% sulfuric acid (13.0 g-130 mmol) indichloromethane (18.0 g-212 mmol). 4-bromobutanol (10.2 g-66.6 mmol) wasthen added to this mixture and the reaction medium was stirred at −5 to5° C. over a period of 2-5 hours. The mixture was poured into cold water(110 g) keeping the temperature between −5° C. and 3° C. Afterdecantation, the upper aqueous phase was extracted with dichloromethaneand the combined organic phases were washed with water, adjusted topH=6-7 by addition of 27.65% sodium hydroxide, washed with brine anddried over magnesium sulfate. Dichloromethane was distilled off undervacuum and crude 4-bromobutyl nitrate (12.7 g-64.1 mmol) was recoveredin a yield of about 96%.

II.3 Preparation of[1R-[1α(Z),2β(R*),3α,5α]]-7-[3,5-dihydroxy-2-(3-hydroxy-5-phenylpentyl)cyclopentyl]-5-heptenoicacid 4-(nitrooxy)butyl ester

Latanoprost acid (97.7%, S-isomer <1%) (213 mg, 0.54 mmol) was dissolvedin 5.0 g anhydrous DMF. K₂CO₃ (206 mg, 1.49 mmol), KI (77 mg, 0.46 mmol)and 4-bromobutylnitrate (805 mg, 25% w/w in methylene chloride, 1.02mmol) were added. The reaction mixture was heated and stirred on arotary evaporator at 45-50° C.

After 1.5 hour, TLC (Si, CH₂Cl₂-MeOH, 5%) showed no starting acid.

The reaction mixture was diluted with 100 ml ethyl acetate, washed withbrine (3×50 ml), dried over MgSO₄ and evaporated to give yellowish oil(420 mg).

¹H NMR/¹³C NMR showed target molecule as a major product together withsome starting 4-bromobutylnitrate and DMF.

HPLC showed no starting acid. Residual solvent, 4-bromobutylnitrate andtarget ester were the main peaks. Butylnitrate ester showed similar UVspectrum as latanoprost and relative retention time was as expected.

Instrument: Bruker 300 MHz Solvent: CDCl₃

¹H-NMR (CDCl₃) δ: 7.29-7.19 (5H, m, Ar); 5.45 (1H, m, CH═CH); 5.38 (1H,m, CH═CH); 4.48 (2H, t, CH ₂—ONO₂); 4.18 (1H, m, CH—OH); 4.10 (2H, t,COOCH ₂); 3.95 (1H, m, CH—OH); 3.68 (1H, m, CH—OH); 2.87-2.60 (2H, m);2.35 (2H, t); 2.25 (2H, m); 2.13 (2H, m); 1.90-1.35 (16H, m).

¹³C-NMR (CDCl₃) ppm: 173.94 (C═O); 142.14; 129.55 (C₅); 129.50 (C₆);128.50; 125.93 78.80 (C₁₁); 74.50 (C₉); 72.70 (C—ONO₂); 71.39 (C₁₅);63.57; 52.99 (Cl₂); 51.99 (C₈); 41.30 (C₁₀); 39.16 (C₁₆); 33.66; 32.21;29.73; 27.04; 26.70; 25.04; 24.91; 23.72; 15.37.

EXAMPLE 2 Synthesis of[1R-[1α(Z),2β(R*),3α,5α]]-7-[3,5-dihydroxy-2-(3-hydroxy-5-phenylpentyl)cyclopentyl]-5-heptenoicacid[2-methoxy-4-[2-propenoyloxy(4-nitrooxybutyl)]]phenylester (compound 11) A) Preparation of Ferulic acid 4-(bromo)butyl ester

To a solution of ferulic acid (1 g, 5.15 mmol) in tetrahydrofurane (40ml), triphenylphosphine (2.7 g, 10.3 mmol) and tetrabromomethane (3.41g, 10.3 mmol) were added. The mixture was stirred at room temperaturefor 4 hours. The mixture was filtered and the solvent was evaporatedunder vacuum. The crude residue was purified by silica gelchromatography, eluent n-hexane/ethyl acetate 7/3. The product (0.77 g)was obtained as a yellow solid. (Yield 46%)

M.p.=83-88° C.

B) Preparation of Ferulic acid 4-(nitrooxy)butyl ester

A solution of compound A (0.8 g, 2.43 mmol) and silver nitrate (1.2 g,7.29 mmol) in acetonitrile (50 ml) was stirred at 40° C., in the dark,for 16 hours. The precipitate (silver salts) was filtered off and thesolvent was evaporated under vacuum. The residue was purified by flashchromatography, eluent n-hexane/ethyl acetate 75/25. The product (0.4 g)was obtained as white powder (yield 53%)

M.p.=63-64° C.

C) Preparation off1R-[1α(Z),2β(R*),3α,5α]-7-[3,5-dihydroxy-2-(3-hydroxy-5-phenylpentyl)cyclopentyl]-5-heptenoicacid [2-methoxy-4-[2-propenoyloxy(4-nitrooxybutyl)]]phenyl ester

To a solution of latanoprost acid (0.2 g, 0.51 mmol) in drytetrahydrofuran (10 ml), in atmosphere inert, ferulic acid4-(nitrooxy)butyl ester (0.32 g, 1.02 mmol) and DMAP (cat. amount) wereadded. The reaction was cooled at 0° C. and EDAC (0.14 g, 0.76 mmol) wasadded. The reaction was stirred at room temperature for 24 hours. Thesolution was treated with water and chloroform, the organic layers wereanidrified with sodium sulfate and concentrated under reduced pressure.The residue was purified by flash chromatography, eluent n-hexane/ethylacetate 3/7. The product (0.2 g) was obtained.

¹H-NMR (CDCl₃) δ: 7.55 (1H, d, CH═CHCO); 7.30-7.03 (8H, m, Ar); 6.35(1H, d, CH═CHCO); 5.48 (2H, m, CH═CH); 4.52 (2H, t, CH ₂—ONO₂); 4.25(2H, t, COO—CH ₂); 4.17 (1H, m, CH—OH); 3.95 (1H, m, CH—OH); 3.85 (3H,s, OCH ₃); 3.65 (1H, m, CH—OH); 2.75 (2H, m); 2.61 (2H, t); 2.48-2.20(5H, m); 1.9-1.20 (19H, m).

¹³C-NMR (CDCl₃): ppm: 171.62 (C═O); 166.69 (C═O); 151.40; 144.36;142.04; 141.55; 133.21; 129.62; 129.41; 128.40; 125.85, 123.27; 121.27;117.96; 111.32; 78.81; 74.84; 72.64 (C—ONO₂); 71.32; 63.61; 55.94;52.99; 51.91; 42.54; 39.08; 35.79; 33.37; 32.12; 29.68; 27.03; 26.53;25.09; 24.90; 23.73.

EXAMPLE 3 Synthesis of[1R-[1α(Z),2β(R*),3α,5α]]-7-[3,5-dihydroxy-2-(3-hydroxy-5-phenylpentyl)cyclopentyl]-5-heptenoicacid 3-(nitrooxymethyl)phenyl ester (compound 4) 1. Preparation of3-[(Bromo)methyl]phenol

3-[(Hydroxy)methyl]phenol was dissolved in acetonitrile (300 ml) anddichloromethane (900 ml) and the resulting mixture was poured in theflask kept under argon; magnetic stirring was set on. The solution wasthen cooled with an ice bath and carbon tetrabromide andtriphenilphosphine were added. The latter was added in small portions inorder to maintain the temperature at ca. 2-3° C.

The solution was stirred for 1 hour at 2-3° C. and then for anadditional hour at room temperature.

After this period the reaction conversion (checked by TLC, usingEtOAc/Petroleum ether 3/7 as the eluent) was complete. The obtainedmixture was evaporated under reduce pressure and 500 ml of petroleumether and 500 ml of EtOAc were added to the resulting yellow thick oilin a 2 l round flask. A pitchy solid was formed. The mixture was keptunder stirring at room temperature overnight and subsequently filteredand concentrated under reduce pressure, furnishing ca. 50 g of an oilyresidue. The oil was purified by flash chromatography over 600 g ofsilica gel, using EtOAc/Petroleum ether 2/8 as the eluent. Furtherpurification was achieved by crystallising the resulting bromide frompetroleum ether. A white solid was obtained (24 g, 64%).

Analysis

TLC: (EtOAc/Petroleum ether 3/7) Rf=0.4

HPLC purity: >98%

FT-IR (KBr, cm⁻¹): 3252, 1589, 1479, 1392, 1270, 1208, 1155, 952, 880,791, 741, 686.

2. Preparation of 3-[(Nitrooxy)methyl]phenol

3-[(Bromo)methyl]phenol was dissolved in 30 ml of acetonitrile andpoured in the flask, kept far from light sources at 0-5° C. under argon;magnetic stirring was set on. Silver nitrate was then added under theseconditions, maintaining the temperature under 5° C. The reaction coursewas followed by TLC (EtOAc/Petroleum ether 3/7 as the eluent). After 4hours and 30 minutes the conversion was complete. The reaction mixturewas then filtered, the precipitated solid was washed with Et₂O and thefiltrate was separated in two batches. The first batch (15 ml) was keptunder argon and in acetonitrile solution at −20° C. The second batch (15ml) was worked-up as follows. The acetonitrile solution was concentratedunder reduce pressure and the resulting oil was dissolved indichloromethane (15 ml) and washed with brine (15 ml). The organic phasewas separated and the aqueous phase was extracted twice withdichloromethane (2×25 ml). The combined organic phases were then driedover MgSO₄, filtered and evaporated. The residue was purified by flashchromatography over 40 g of silica gel using EtOAc/Petroleum ether 2/8as the eluent. The nitrate was obtained as an oil (0.6 g, 67%).

Analysis

TLC: (EtOAc/Petroleum ether 3/7) Rf=0.35

HPLC purity: >98%

MS (ESI−): 168 (M⁺−1)

FT-IR (neat oil, cm⁻¹): 3365, 1632, 1599, 1459, 1282, 1160, 923, 867,793, 757.

¹H NMR (CDCl₃, 300 MHz) δ 5.31 (2H, s), 5.45 (1H, br s), 6.78-6.84 (2H,m), 6.87-6.92 (1H, m), 7.17-7.24 (1H, m).

3. Preparation of[1R-[1α(Z),2β(R*),3α,5α]]-7-[3,5-dihydroxy-2-(3-hydroxy-5-phenylpentyl)cyclopentyl]-5-heptenoicacid 3-(nitrooxymethyl)phenyl ester

To a solution of latanoprost acid (0.11 g, 0.28 mmol) in chloroform (20ml), in atmosphere inert, 3-(nitrooxymethyl)phenol (0.01 g, 0.56 mmol)and DMAP (cat. amount) were added. The reaction was cooled at 0° C. andEDAC (0.08 g, 0.42 mmol) was added. The reaction was stirred at roomtemperature for 24 hours. The solution was treated with water, theorganic layers were anidrified with sodium sulfate and concentratedunder reduced pressure. The residue was purified by flashchromatography, eluent n-hexane/ethyl acetate 3/7. The product (0.1 g)was obtained.

¹H-NMR (CDCl₃) δ: 7.41 (1H, t, Ar); 7.31-7.10 (8H, m, Ar); 5.48 (2H, m,CH═CH); 5.43 (2H, s, CH ₂—ONO₂); 4.16 (1H, m, CH—OH); 3.95 (1H, m,CH—OH); 3.65 (1H, m, CH—OH); 2.75 (2H, m); 2.61 (2H, t); 2.48-2.20 (5H,m); 1.9-1.20 (11H, m).

EXAMPLE 4 Synthesis of[1R-[1α(Z),2β(R*),3α,5α]]-7-[3,5-dihydroxy-2-(3-hydroxy-5-phenylpentyl)cyclopentyl]-5-heptenoicacid 4-(nitrooxymethyl)benzyl ester (compound 9) A)[1R-[1α(Z),2β(R*),3α,5α]]-7-[3,5-dihydroxy-2-(3-hydroxy-5-phenylpentyl)cyclopentyl]-5-heptenoicacid 4-(bromomethyl)benzyl ester

To a solution of latanoprost acid (0.5 g, 1.2 mmol) in chloroform (50ml), in inert atmosphere, 4-(bromomethyl)benzyl alcohol (0.4 g, 1.92mmol) and DMAP (cat. amount) were added. The reaction was cooled at 0°C. and EDAC (0.37 g, 1.92 mmol) was added. The reaction was stirred atroom temperature for 5 hours. The solution was treated with water, theorganic layers were anidrified with sodium sulfate and concentratedunder reduced pressure. The residue was purified by flashchromatography, eluent n-hexane/ethyl acetate 3/7. The product (0.47 g)was obtained.

B)[1R-[1α(Z),2β(R*),3α,5α]]-7-[3,5-dihydroxy-2-(3-hydroxy-5-phenylpentyl)cyclopentyl]-5-heptenoicacid 4-(nitrooxymethyl)benzyl ester

A solution of compound A (0.4 g, 0.7 mmol) and silver nitrate (0.23 g,1.4 mmol) in acetonitrile (50 ml) was stirred at 40° C., in the dark,for 4 hours. The precipitated (silver salts) was filtered off and thesolvent was evaporated under vacuum. The residue was purified by flashchromatography, eluent n-hexane/ethyl acetate 7/3. The product (0.15 g)was obtained as oil.

¹H-NMR δ: 7.39 (4H, s, Ar); 7.31-7.17 (5H, m, Ar); 5.44 (2H, m, CH═CH);5.42 (2H, s, CH₂—ONO₂); 5.30 (2H, s, O—CH ₉—Ar); 4.15 (1H, m, CH—OH);3.95 (1H, m, CH—OH); 3.67 (1H, m, CH—OH); 2.75 (2H, m); 2.41 (2H, t);2.48-1.20 (16H, m).

EXAMPLE 5 Synthesis of[1R-[1α(Z),2β(R*),3α,5α]]-7-[3,5-dihydroxy-2-(3-hydroxy-5-phenylpentyl)cyclopentyl]-5-heptenoicacid 3-(nitrooxy)propyl ester (compound 78)

The compound is synthesized using the procedure described in EXAMPLE 4starting from latanoprost acid and 3-bromopropanol.

EXAMPLE 6 Synthesis of[1R-[1α(Z),2β(R*),3α,5α]]-7-[3,5-dihydroxy-2-(3-hydroxy-5-phenylpentyl)cyclopentyl]-5-heptenoicacid 2-(nitrooxy)ethyl ester (compound 77)

The compound is synthesized using the procedure described in EXAMPLE 4starting from latanoprost acid and 2-bromoethanol.

EXAMPLE 7 Synthesis of[1R-[1α(Z),2β(R*),3α,5α]]-7-[3,5-dihydroxy-2-(3-hydroxy-5-phenylpentyl)cyclopentyl]-5-heptenoicacid 6-(nitrooxy)hexyl ester (compound 79)

The compound is synthesized using the procedure described in EXAMPLE 4starting from latanoprost acid and 6-bromohexanol.

EXAMPLE 8 Synthesis of[1R-[1α(Z),2β(R*),3α,5]]-7-[3,5-dihydroxy-2-(3-hydroxy-5-phenylpentyl)cyclopentyl]-5-heptenoicacid 2-(nitrooxy)-1-methylethyl ester (compound 80)

The compound is synthesized using the procedure described in EXAMPLE 4starting from latanoprost acid and 1-bromo-2-propanol.

EXAMPLE 9 Synthesis of[1R-[1α(Z),2β(R*),3α,5α]]-7-[3,5-dihydroxy-2-(3-hydroxy-5-phenylpentyl)cyclopentyl]-5-heptenoicacid 2-(nitrooxy)propyl ester (compound 81)

The compound is synthesized using the procedure described in EXAMPLE 4starting from latanoprost acid and 2-chloro-1-propanol.

EXAMPLE 10 Synthesis of[1R-[1α(Z),2β(R*),3α,5α]]-7-[3,5-dihydroxy-2-(3-hydroxy-5-phenylpentyl)cyclopentyl]-5-heptenoicacid 2-(nitrooxy)-1-(nitrooxymethyl)ethyl ester (compound 82)

The compound is synthesized using the procedure described in EXAMPLE 4starting from latanoprost acid and 1,3-dibromo-2-propanol.

EXAMPLE 11 Synthesis of[1R-[1α(Z),2β(R*),3α,5α]]-7-[3,5-dihydroxy-2-(3-hydroxy-5-phenylpentyl)cyclopentyl]-5-heptenoicacid [2-methoxy-4-[2-propenoyloxy(2-nitrooxyethyl)]]phenyl ester(compound 83)

The compound is synthesized using the procedure described in EXAMPLE 2starting from latanoprost acid and ferulic acid 2-(nitrooxy)ethyl ester.

EXAMPLE 12 Synthesis of[1R-[1α(Z),2β(R*),3α,5α]]-7-[3,5-dihydroxy-2-(3-hydroxy-5-phenylpentyl)cyclopentyl]-5-heptenoicacid 2-methoxy-4-[2-propenoyloxy(3-nitrooxmethylphenyl)]]phenyl ester(compound 84)

The compound is synthesized using the procedure described in EXAMPLE 2starting from latanoprost acid and ferulic acid 3-(nitrooxymethyl)phenylester.

EXAMPLE 13 Synthesis of[1R-[1α(Z),2β(R*),3α,5α]]-7-[3,5-dihydroxy-2-(3-hydroxy-5-phenylpentyl)cyclopentyl]-5-heptenoicacid 2-methoxy-4-[2-propenoyloxy(4-nitrooxmethylbenzyl)]]phenyl ester(compound 85)

The compound is synthesized using the procedure described in EXAMPLE 2starting from latanoprost acid and ferulic acid 4-(nitrooxymethyl)benzylester.

EXAMPLE 14 Synthesis of[1R-[1α(Z),2β(R*),3α,5α]]-7-[3,5-dihydroxy-2-(3-hydroxy-5-phenylpentyl)cyclopentyl]-5-heptenoicacid (4-nitrooxmethyl)phenyl ester (compound 6)

The compound is synthesized using the procedure described in EXAMPLE 4starting from latanoprost acid 4-(chloromethyl)phenyl ester.

EXAMPLE 15 Synthesis of[1R-[1α(Z),2β(R*),3α,5α]]-7-[3,5-dihydroxy-2-(3-hydroxy-5-phenylpentyl)cyclopentyl]-5-heptenoicacid (3-nitrooxmethyl)benzyl ester (compound 8)

The compound is synthesized using the procedure described in EXAMPLE 4starting from latanoprost acid 4-(bromomethyl)benzyl ester.

EXAMPLE 16 Preparation of an Ophthalmic Composition Using[1R-[(1α(Z),2α(R*),3α,5α]]-7-[3,5-dihydroxy-2-(3-hydroxy-5-phenylpentyl)cyclopentyl]-5-heptenoicacid 4-(nitrooxy)butyl ester (compound 1)

Ingredient Amount (mg/ml) Compound 1 0.1 Tween 80 5 Benzalkoniumchloride 0.2 Buffer q.s. Buffer: NaCl 4.1 mg/ml NaH₂PO₄ (anh.) 4.74mg/ml NaH₂PO₄ (monohyd.) 4.6 mg/ml water for injection qs.

EXAMPLE 17 Evaluation of Nitric Oxide-Mediated Activity

The formation of cyclic guanosine-3′,5′ monophosphate (cGMP) in cells inthe eye is involved in the regulation of aqueous humor flow. Thus,elevation of cGMP levels leads to decreased aqueous humor production andreduction of intraocular pressure.

We measured the effects of test drugs on cGMP formation in a wellestablished cell assay.

Undifferentiated pheochromocytoma cells (PC12) were used. The monolayercells were incubated for 45 min in Hank's Balanced Salt Solutionenriched with 10 mM Hepes, 5 mM MgCl₂ and 0.05% ascorbic acid at thefinal pH of 7.4 and containing 100 μM of the phosphodiesteraseinhibitor, isomethyl-butyl-xanthine (IBMX), 30 μM of the guanylylcyclase inhibitor, YC-1, and the test drugs at the appropriateconcentration. The reaction was terminated by the removal of theincubating buffer followed by the addition of 50 L of 100% ice-coldethanol. The plate was then dried under hot air steam and the residuedissolved, extracted and analysed using commercially available cycliccGMP enzyme immunoassay kit.

The results are reported in Table 1. The concomitant application ofdifferent concentrations of the various Latanoprost nitroderivatives(1-50 μM) elicited cGMP accumulation in a concentration-dependentfashion.

These effects were not shared by the parent drug Latanoprost suggestingthat such effects are dependent on the release of exogenous NO.

TABLE 1 Potency and Efficacy of Latanoprost and respectivenitroderivatives on cGMP accumulation in rat pheochromocytoma cells.EC₅₀ E_(max) Drugs (μM) (% over vehicle) Latanoprost Not effective Noteffective Compound 1 (ex. 1) 2.4 290 Compound 4 (ex. 3) 4.4 450 Compound11 (ex. 2) 1.5 480 EC₅₀ = effective concentration producing half maximalresponse E_(max) = maximum effect

EXAMPLE 18 Evaluation of the Efficacy of Latanoprost Nitroderivative onIntraocular Pressure

Male NZW rabbits ranging from 3-5 kgs of body weight were used in thisstudy. Briefly, the ability of Latanoprost nitroderivative (compound 4,EXAMPLE 3) at reducing intraocular pressure (IOP) was tested in animalspreviously treated with intracameral injection of 0.25% carbomersolution installation until after a stable increase of the intraocularpressure was reached. In this particular study, test drugs wereadministered to one eye with the dosage schedule of 1 drop/eye/day for 5days a week with a physiologic solution containing 0.005% of control ortest compounds. The IOP was monitored 3 h after drug application,two-three times weekly for a total of 4 weeks. This concentration waschosen as it reflects that of latanoprost isopropyl ester currently usedin clinic to treat the increase of IOP observed in glaucoma patients.Furthermore, at each visit, about 200 μl of aqueous humor was collectedusing a 30 gauge needle from both eyes under lidocaine anesthesia forfurther biochemical evaluation of cGMP, camp and nitrite/nitratecontents.

The installation of 0.25% carbomer solution into the eye resulted in aprofound increase of the IOP to about 40 mmHg that remained stablethereafter. However, the administration of the compound 4 (EX. 3) withthe dose schedule outline in the method session, decreased theintraocular pressure of these animals of about 50% within 7 days ofrepeated treatments and over 65% by the end of the study (See Table 2).In contrast, neither Latanoprost acid (data not shown) nor its isopropylderivative elicited any appreciable change (see Table 2). Given theliterature available documenting that Latanoprost is virtually noteffective in rabbits, the observed effects are likely to be attributedto the presence of the nitric oxide (NO) moiety onto Latanoprostnitroderivative rather than the parent compound.

Biochemical measurements of cGMP, cAMP and NOx in the intraocularaqueous humor further supported the role of NO at decreasing the IOP ofthese animals. In fact, as shown in Table 3, the extent of cGMP and NOxincreased following the application of the compound 4 (EX. 3) over the4-week treatment. These effects turn out to be highly specific as theamount of intraocular cAMP remained unaltered in these animals.Latanoprost isopropyl ester did not significantly affect the levels ofeither cGMP, cAMP or nitrites when given at equimolar doses to that ofthe respective nitroderivative (see Table 3).

TABLE 2 Reversal of stimuli carbomer-evoked increase in IOP before(pre-treatment) and after eye-installation of equimolar Latanoprostisopropyl ester or the respective nitroderivative IOP Pre- mmHgtreatment* Day 2 Day 7 Day 10 Day 15 Day 17 Day 23 Day 25 Latanoprost 37± 2 34 ± 2 33 ± 3 30 ± 1 31 ± 2 30 ± 2 32 ± 2 30 ± 2 isopropyl esterCompound 4 42 ± 2 31 ± 1 26 ± 1 20 ± 1 18 ± 2 16 ± 1 15 ± 1 14 ± 1 (ex.3) *Pre-treatment values correspond to baseline IOP evoked following theintracameral installation of 0.25% carbomer solution.

TABLE 3 Effects of Latanoprost isopropyl ester and the respectivenitroderivative on cGMP, cAMP and NOx content in carbomer-treatedrabbits. IOP Pre- I II III IV mmHg treatment* Week Week Week Week cGMP(fmol/mg prot) Latanoprost isopropyl 87 ± 6 88 ± 6 98 ± 6 99 ± 6 100 ±6  ester Compound 4 (ex. 3) 88 ± 5 102 ± 5  125 ± 5  140 ± 5  160 ± 5 cAMP (fmol/mg prot) Latanoprost isopropyl 510 ± 18 550 ± 22 600 ± 30 620± 31 625 ± 31 ester Compound 4 (ex. 3) 520 ± 20 600 ± 25 650 ± 31 680 ±28 660 ± 22 NOx (nmol/mg prot) Latanoprost isopropyl 16 ± 1 18 ± 2 18 ±1 19 ± 2 19 ± 2 ester Compound 4 (ex. 3) 17 ± 1 22 ± 2 25 ± 3 26 ± 3 28± 3 *Pre-treatment values correspond to baseline IOP evoked followingthe intracameral installation of 0.25% carbomer solution.

1.-20. (canceled)
 21. A compound of the formula

or a pharmaceutically acceptable salt or stereoisomer thereof.
 22. A pharmaceutical composition comprising a compound of the formula

or a pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable carrier.
 23. A pharmaceutical composition according to claim 22 in a suitable form for topical administration.
 24. A pharmaceutical composition according to claim 22, wherein said compound is administered as a solution, suspension or emulsion in an ophthalmically acceptable vehicle.
 25. A method for treating glaucoma or ocular hypertension, said method comprising contacting an effective intraocular pressure reducing amount of a pharmaceutical composition according to claim 23 with the eye in order to reduce eye pressure and to maintain said pressure on a reduced level.
 26. A method for treating glaucoma or ocular hypertension, said method comprising contacting an effective intraocular pressure reducing amount of a pharmaceutical composition according to claim 24 with the eye in order to reduce eye pressure and to maintain said pressure on a reduced level.
 27. (canceled)
 28. (canceled) 